Dataset Information

Mass spectrometric characterization of HSV-1 L-particles from human dendritic cells and BHK21 cells and analysis of their functional role

ABSTRACT: Herpes simplex virus type 1 (HSV-1) is a very common human pathogenic virus among the world’s 12 population. The lytic replication cycle of HSV-1 is, amongst others, characterized by a tripartite viral 13 gene expression cascade, the assembly of nucleocapsids involving their subsequent nuclear egress, 14 tegumentation, re-envelopment and the final release of progeny viral particles. During productive 15 infection of a multitude of different cell types, HSV-1 generates not only infectious heavy (H-) 16 particles, but also non-infectious light (L-) particles, lacking the capsid. In monocyte-derived mature 17 dendritic cells (mDCs), HSV-1 causes a non-productive infection with the predominant release of L-18 particles. Until now, the generation and function of L-particles is not well understood, however, they 19 are described as factors transferring viral components to the cellular microenvironment. To obtain 20 deeper insights into the L-particle composition, we performed a mass-spectrometry-based analysis of 21 L-particles derived from HSV-1-infected mDCs or BHK21 cells and H-particles from the latter one. 22 In total, we detected 63 viral proteins in both H- and L-particle preparations derived from HSV-1-23 infected BHK21 cells. In L-particles from HSV-1-infected mDCs we identified 41 viral proteins 24 which are differentially distributed compared to L-particles from BHK21 cells. In this study, we 25 present data suggesting that L-particles modify mDCs and suppress their T cell stimulatory capacity. 26 Due to the plethora of specific viral proteins incorporated into and transmitted by L-particles, it is 27 tempting to speculate that L-particles manipulate non-infected bystander cells for the benefit of the 28 virus.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Mesocricetus Auratus (golden Hamster)

TISSUE(S): Dendritic Cell

DISEASE(S): Herpes Simplex

SUBMITTER: Joerg Hofmann

LAB HEAD: Alexander Steinkasserer

PROVIDER: PXD020845 | Pride | 2021-09-09

REPOSITORIES: Pride

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Mass Spectrometric Characterization of HSV-1 L-Particles From Human Dendritic Cells and BHK21 Cells and Analysis of Their Functional Role.

Birzer Alexandra A Kraner Max Edmund ME Heilingloh Christiane Silke CS Mühl-Zürbes Petra P Hofmann Jörg J Steinkasserer Alexander A Popella Linda L

Frontiers in microbiology 20200929

Herpes simplex virus type 1 (HSV-1) is a very common human pathogenic virus among the world's population. The lytic replication cycle of HSV-1 is, amongst others, characterized by a tripartite viral gene expression cascade, the assembly of nucleocapsids involving their subsequent nuclear egress, tegumentation, re-envelopment and the final release of progeny viral particles. During productive infection of a multitude of different cell types, HSV-1 generates not only infectious heavy (H-) particle ...[more]

PMID: 33117298

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Project description:The overall goal of the study was to use in vivo data combined with functional genomics to define gene expression signatures representative of a spectrum of HSV CNS infections. Innate immune deficiencies result in a spectrum of severe clinical outcomes following infection. In particular, there is a strong association between loss of the signal transducer and activator of transcription (Stat) pathway, breach of the blood-brain barrier (BBB), and virus-induced neuropathology. The gene signatures that characterize resistance, disease, and mortality in the virus-infected nervous system have not been defined. Herpes simplex virus type 1 (HSV-1) is commonly associated with encephalitis in humans, and humans and mice lacking Stat1 display increased susceptibility to HSV central nervous system (CNS) infections. In this study, two HSV-1 strains were used, KOS (wild type [WT]), and Δvhs, an avirulent recombinant lacking the virion host shutoff (vhs) function. In addition, two mouse strains were used: strain 129 (control) and a Stat1-deficient (Stat1(-/-)) strain. Using combinations of these virus and mouse strains, we established a model of infection resulting in three different outcomes: viral clearance without neurological disease (Δvhs infection of control mice), neurological disease followed by viral clearance (Δvhs infection of Stat1(-/-) mice and WT infection of control mice), or neurological disease followed by death (WT infection of Stat1(-/-) mice). Through the use of functional genomics on the infected brain stem and liver, we determined gene signatures that were representative of the three infection outcomes. Gender matched, 6- to 8- week old immunocompetent, control 129S6 and 129S6 Stat1 knockout mice were infected corneally with 2x10^6 PFU of either wild type HSV-1, a vhs-null HSV virus, or mock-infected. Brain stems and liver of individual mice were isolated at days 1, 3, 5 and 7 post-inoculation for microarray analysis. For microarray analysis, samples were collected from n=2 animals (1 male, 1 female) per mouse strain and virus strain for each time point. Equal masses of tissue were pooled from two mock-infected mice per time point and run on microarray.

Project description:For Samples 1-8 and 11-18: The innate immune sensor retinoic acid-inducible gene-I (RIG-I) detects double-stranded RNA derived from RNA viruses, and recent studies have demonstrated that RIG-I also plays a role in the antiviral response to DNA viruses. To identify the physiological RNA species that are recognized by RIG-I during HSV-1 infection, we purified the RNAs that co-immunoprecipitated with FLAG-tagged RIG-I in transfected human embryonic kidney (HEK) 293T cells that had been infected with a recombinant HSV-1 (hereafter referred to as HSV-1 mut) containing a mutation (K220A) in the viral serine/threonine protein kinase US3 that abolishes its catalytic activity, as the viral kinase is known to antagonize type-I IFN responses. As controls, RNA species bound to FLAG-RIG-I in uninfected cells and RNA bound to FLAG-GFP from both HSV-1 mut-infected and uninfected cells were also purified. RIG-I-bound RNA and total RNA extracted from uninfected and HSV-1 mut-infected cells were analyzed by RNAseq, and the resulting sequences were mapped to both the HSV-1F-strain and human genome (hg38). This analysis revealed that several human transcripts were highly enriched in the RIG-I-bound fraction from infected cells; in contrast, the enrichment of viral sequences was low. The cellular transcripts that were most abundant in the RIG-I fraction were predominantly non-coding RNAs from different subclasses, as well as some coding RNAs. For Samples 9 and 10: HSV-1 infection is known to induces changes in the transcriptional profile of the infected cell. To analyze global changes in RNA transcript levels in infected cells, total RNA was extracted from HEK 293T cells that were infected with wild-type (WT) HSV-1. For comparison, total RNA was extracted from HEK 293T cells that remained uninfected. Next, RNAseq analysis was performed. The resulting sequences were mapped to the human genome, and gene inductions were calculated and normalized to uninfected samples to determine changes in gene expression upon infection.

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